Exposure to environmental stress such as radiation, poor nutrition or smoking, can cause chromosome breaks, the most hazardous lesion formed by radiation. The micronucleus test is an established method to analyze in vivo chromosomal damage and has been used by many investigators to monitor DNA integrity following exposure to poor nutrition, toxicants and other stressors. The test is based on the observation that a secondary nucleus (micronucleus) is formed around a chromosomal fragment, outside the main nucleus of a dividing cell. A micronucleus may also be produced due to a lagging whole chromosome formed as a result of a chromosome loss at anaphase. The measurement of micronuclei (MN) in peripheral blood lymphocytes has been a conventional tool to biomonitor human populations for DNA damage, and is widely used in product development to fulfill regulatory requirements for assessment of chromosomal damage (Fenech, M., Mutat Res (2000) 455:81-95; Fenech, M., Drug Discov Today (2002) 7:1128-1137). The lymphocyte assay applies a chemical to block cytokinesis after a single cell division and the MN are manually counted and scored using microscopy (Xue et al, Int J Cancer (1992) 50:702-705; Fenech et al, Environ Mol Mutagen (1997) 30:112-118). Although this is a low throughput assay it allows detection of other DNA-damage markers, such as nucleoplasmic bridges and nuclear buds and is effective in scoring 1000 cells for effects of folate deficiency and ionizing radiation. The mitogenic response is also indicative of immune responsiveness. However, to reliably detect small increases in MN frequency in people at risk, monitoring of a large number of cells in exposed and control groups is required. To accomplish this, a simple assay that enables scoring of large number of cells in short periods of time should be performed.
A powerful assay in detecting small changes in genome damage in animal models is the mouse in vivo erythrocyte MN test (Mavournin et al., Mutat Res (1990) 239:29-80). Micronuclei are particularly apparent in red blood cells (RBC), which otherwise lack DNA. During erythropoiesis, a hematopoietic stem cell differentiates into an erythroblast, and then expels its nucleus to become a reticulocyte. The newly formed reticulocyte is then released from the bone marrow into the bloodstream, where it develops into a mature erythrocyte. Although the main nucleus is lost during reticulocyte formation, micronuclei are retained in the reticulocyte cytoplasm (Fenech, M., 2000). Dertinger et al have developed an in vivo rodent micronucleus test for detecting micronucleated reticulocytes by flow cytometry using fluorescent labels for transferrin receptor (CD71) and fluorescent DNA stains such as propidium iodide (Mutat Res (1996) 371:283-292).
In humans, in contrast to mice, micronucleated erythrocytes are soon filtered from the circulating blood by the spleen and therefore are not generally available for analysis. Abramsson-Zetterberg et al have described a method to measure MN in an enriched peripheral-blood reticulocyte-population (Environ Mol Mutagen (2000) 36:22-31). With this method, Hoechst 33342 and thiazole orange were used to stain DNA and RNA, respectively. They showed that MN frequency in reticulocytes approximate those observed in bone marrow. However their method includes multiple laborious steps and required the use of a dual-laser flow cytometer with a UV laser to excite the Hoechst 33342 fluorochrome. This type of flow cytometer is more specialized and not widely available in common laboratory settings. Similar to the in vivo rodent micronucleus test, Dertinger et al. further improved the scoring of micronucleated reticulocytes in humans to enable the use of widely available bench top instruments (Mutat Res (2002) 515:3-14; Mutat Res (2003) 542:77-87). However, their procedure required long data-collection times.
We have developed a simple method to isolate and analyze immature reticulocytes in the peripheral blood for the presence of micronuclei that is useful in establishing the relation between environmental stress (e.g. micronutrient deficiencies) and chromosomal damage. This method enables rapid analysis of large numbers of cells by applying single-laser flow-cytometry to measure micronuclei in an enriched transferrin-positive reticulocyte population.